OP0235 GENOME-WIDE DNA METHYLATION ANALYSIS IN MULTIPLE IMMUNE CELL POPULATIONS REVEALS WIDESPREAD PROMOTER AND INTERGENIC B CELL HYPERMETHYLATION IN SYSTEMIC LUPUS ERYTHEMATOSUS
Authors: C. Ulff-Møller, J. Tost, S. Jacobsen
Journal: Annals of the Rheumatic Diseases (2022)

Background Patients with systemic lupus erythematosus (SLE) display an aberrant DNA methylation (DNAm) pattern with a predominant interferon signature. However, the DNAm patterns in purified immune cell populations are not well described. Objectives To examine genome-wide DNAm changes in sorted CD4 + T cells, monocytes, granulocytes and B cells in SLE patients compared to healthy controls (HC). Methods Genome-wide analysis was performed in 20 SLE patients with active lupus nephritis (LN) and 20 age-, sex- and ethnicity-matched HCs. Peripheral blood was processed using gradient density centrifugation for the granulocyte fraction, and CD4 + T-cells, monocytes and B cells were isolated from the mononuclear cell fraction using a RoboSep device (Stemcell Technologies). DNA was extracted using the DNA/RNA/miRNA Universal kit (Qiagen). Genome-wide DNAm was evaluated using Infinium MethylationEPIC BeadChips (Illumina Inc.). Top differentially methylated CpG sites (DMC) were validated using pyrosequencing, and further analysed in follow-up samples, as well as extended to another 36 SLE patients with inactive LN, 36 SLE with never-LN and 20 HCs. Patients and HCs were compared using a paired t -test stratified by cell type. A false discovery rate (FDR) &lt;0.05 was considered statistically significant. Clustering of non-gene annotated CpGs was defined as distance &lt;5000 bp with proximity to neighbouring genes &lt;1x10 5 bp, identified through mapping of Entrez Gene Identifiers. Results Overall, SLE patients with active LN compared to HCs exhibited DMCs in 22, 46, 312 and 78,068 probes in CD4 + T cells, monocytes, granulocytes and B cells, respectively. In CD4 + T cells, monocytes and granulocytes, the majority of DMCs were hypomethylated and related to interferon-regulated genes. In B cells, the majority of DMCs were hypermethylated with 1360 genes in the promoter region restricted to differential methylation &gt;10% and ≥2 DMCs, of which 1087 were hypermethylated. Of these, the most significantly hypermethylated CpGs in the promoter region included CABYR, TMPRSS7, SLC6A2, KLK10/KLK11, WIZ, LPAR1, ZNF385B and IL1R1 (Figure 1). Top hypomethylated genes included IFI44L and MX1 . Ingenuity Pathway Analysis (IPA) predicted top upstream regulators in B cells to be IL4 , TNF and IFNG . In addition, we identified 1437 DMCs associated with ncRNA and 4626 DMCs with no genic association. The non-gene associated DMCs were related to 275 non-coding clusters. Top non-coding clusters were in proximity to BMP7 , ARHGEF10 , PRDM1, RIN2 and CCR6, which themselves were differentially methylated. IPA predicted IL2, CD40LG and NFKB1 as the most important upstream regulators for neighbouring genes of non-coding clusters. Pyrosequencing confirmed B cell hypermethylation in CXCR5, DDR1, MTA3, RAB30 and TRAF5, and in non-coding clusters located on chromosome 3 upstream of FGF12 and chromosome 22 downstream of XPNPEP3 and RBX1 . Hypermethylation in CXCR5, DDR1, MTA3, RAB30 and hypomethylation in IFI44L, LGALS3BP and PARP9 was stable at follow-up as well as in patients with inactive and never-LN. Patients in the active LN group exhibited significantly more pronounced hypomethylation in PLSCR1 in CD4 + T-cells, granulocytes and B-cells compared to the other patient groups. Greater β2 microglobulin, anti-DNA antibodies and disease activity were significantly associated with greater B cell CXCR5 hypermethylation (p&lt;0.001), IFI44L hypomethylation in all but CD4 + T cells , LGALS3BP hypomethylation in granulocytes (p&lt;0.0001) , PARP9 hypomethylation in all but B cells and PLSCR1 hypomethylation in all but monocytes. Proteinuria was related to PLSCR1 hypomethylation in all but monocytes, as well as hypermethylation at the non-coding cluster in chromosome 3 (p=0.05) and MTA3 (p=0.03) in B cells. Conclusion B cell hypermethylation in SLE patients is widespread, and may indicate a novel mechanism for SLE pathogenesis. Differential methylation of several interferon-regulated genes may be associated with disease activity. Disclosure of Interests None declared